An In vitro Study of Bio-Control and Plant Growth Promotion Potential of Salicaceae Endophytes

Abstract

Microbial communities in the endosphere of Salicaceae plants, poplar (Populus trichocarpa) and willow (Salix sitchensis), have been demonstrated to be important for plant growth promotion, protection from biotic and abiotic stresses, and degradation of toxic compounds. Our study aimed to investigate bio-control activities of Salicaceae endophytes against various soil borne plant pathogens including Rhizoctonia solani AG-8, Fusarium culmorum, Gaeumannomyces graminis var. tritici, and Pythium ultimum. Additionally, different plant growth promoting traits such as biological nitrogen fixation (BNF), indole-3-acetic acid (IAA) biosynthesis, phosphate solubilization, and siderophore production were assessed in all bio-control positive strains. Burkholderia, Rahnella, Pseudomonas, and Curtobacterium were major endophyte genera that showed bio-control activities in the in-vitro assays. The bio-control activities of Burkholderia strains were stronger across all tested plant pathogens as compared to other stains. Genomes of sequenced Burkholderia strains WP40 and WP42 were surveyed to identify the putative genes involved in the bio-control activities. The ocf and hcnABC gene clusters responsible for biosynthesis of the anti-fungal metabolites, occidiofungin and hydrogen cyanide, are present in the genomes of WP40 and WP42. Nearly all endophyte strains showing the bio-control activities produced IAA, solubilized tricalcium phosphate, and synthesized siderophores in the culture medium. Moreover, some strains reduced acetylene into ethylene in the acetylene reduction assay, a common assay used for BNF. Salicaceae endophytes could be useful for bio-control of various plant pathogens, and plant growth promotion possibly through the mechanisms of BNF, IAA production, and nutrient acquisition.

Keywords: Burkholderia; Salicaceae endophytes; bio-control; soil borne plant pathogens.

Figure 1

In-vitro inhibition of R. solani AG-8 (Plate A), and Ggt (Plate B) by poplar endophytes; WP40 and WP42 respectively; with reference to inhibition of Ggt by the positive control strain, Pf 2-79 (Plate C).

Figure 2

Annotation and graphical representation of the ocf gene cluster in WP42 [Locus tags: EX20DRAFT_04106 (ambR1)–04091 (ocfN)] and WP40 [Locus tags: Ga0008008_11691 (ambR1)–116106 (ocfN)]. The same colors were used for open reading frames (arrow) encoding for proteins with the same function (arrows). The right pane shows the protein domain.

Figure 3

Protein alignment of putative hcn biosynthetic genes in WP40 (Locus tag: Ga0008008_101290–101292), and WP42 (Locus Tag: EX20DRAFT_00138–00140) with HcnA, B, and C from P. fluorescens Pf-5. The conserved amino acid residues of Fe-S binding sites, in HcnA, and ADP-binding motif, in HcnB and HcnC, important for hydrogen cyanide synthases function (Laville et al., ; Ryall et al., 2008), are indicated with black triangles.

Figure 4

IAA production by the Salicaceae endophytes. Strains were grown in triplicate for 4 days in TYC broth containing 0.1% L-tryptophan. The IAA amount produced by each endophyte strain was calculated using the standard curve.

Figure 5

Tricalcium phosphate (NBRIP) plates showing the phosphate solubilization gradient: Plate (A); no solubilization, Plate (B); moderate solubilization, and Plate (C); high solubilization.

Figure 6

Area of orange halo (cm²) displayed by different Salicaceae endophytes in CAS agar plates. Histograms that do not share a letter are significantly different at <0.05 probability level.

Figure 7

Annotation of orb and pvd genes in WP42 [Locus tags: EX20DRAFT_02566(orbS)–02579 (orbL)], and WP40 [Locus tags: Ga0008008_110136 (orbS)–110149 (orbL)], and B. cepacia groups.

Figure 8

Acetylene reduction assay. Ethylene produced by endophyte strains after 24 h of exposure to acetylene. Histograms that do not share a letter are significantly different at <0.05 probability level.